Defined sequence RNA synthesis in the 3′→5′ direction is now well established and currently in use for synthesis and development of a vast variety of therapeutic grade RNA aptamers, tRNAs, siRNA and biologically active RNA molecules. This 3′→5′ synthetic approach utilizes 3′-amidites and 3′-supports to lead to oligonucleotides.

However, the 3′→5′ synthetic approach has a major draw back of the migration of the O-TBDMS group from the 3′-position to the 2′-position, and vice versa:

In contrast, the 5′→3′ synthetic approach, which is also called the “reverse direction” synthesis, or the “reverse synthesis”, has successfully circumvented the issue of the migration of the O-TBDMS group. The 5′→3′ synthetic approach utilizes 5′-amidites and 5′-supports to lead to oligonucleotides, and can be summarized as:

The 5′→3′ approach for RNA synthesis is documented in U.S. Pat. No. 8,309,707 and U.S. Pat. No. 8,541,569. By the 5′→3′ approach, RNA's of various lengths have been successfully achieved, such as 31-mer, 43-mer, 74-mer and 76-mers. For instance:
A. 31-mer G-rich RNA Chimera Synthesis (Oligonucleotide containing 16 Guanosines) (see FIG. 1). The sequence is: 5′-ACG GGA AGA GGG AAmeU GAG GGmeU ACG AGG GCGme U-3′ (SEQ. ID No. 4). Please note that “meU” is the modified base 2′-O-methyluridine.
B. 43-mer RNA Synthesis (see FIG. 2). The sequence is: GGC CCA UCC GUG GAG 988 876 77C CCA GGG 888 767 76C GGU C (SEQ. ID No. 5). Please note that:
“6” represents the modified base 2′-O-methyladenosine;
“7” represents the modified base 2′-O-methylcytidine;
“8” represents the modified base 2′-O-methylguanosine; and
“9” represents the modified base 2′-O-methyluridine.
C. 74-mer RNA Synthesis (see FIG. 3). The sequence is: UCC UCU GUA GUU CAG UCG GUA GAA CGG CGG ACU UUC AAU CCG UAU GUC ACU GGU UCG AGU CCA GUC AGA GGA GC (SEQ. ID No. 6).
D. 76-mer RNA Synthesis (see FIG. 4) The sequence is: GCC CGG AUA GCU CAG UCG GUA GAG CAU CAG ACU UUU UAU CUG AGG GUC CAG GGU UCA AGU CCC UGU UCG GGC GCC A (SEQ. ID No. 7)
However, there is a need to achieve the synthesis of longer RNA, such as 100-mer to 200-mer, especially in the application of the 5′→3′ approach for the RNA synthesis.
Concurrently, several situations have made the research of longer RNA very crucial.
(A) Non-coding RNAs (ncRNAs) are known to regulate mammalian X-chromosome inactivation, and may also be processed to yield small RNAs (http://genesdev.cshlp org/content/23/13/1494.long).
(B) The RNA interference (RNAi) machinery has well-characterized roles in generation of microRNAs (miRNAs) and small interfering RNAs (siRNAs) that regulate gene expression post-transcriptionally. A 2.4-kb unspliced, polyadenylated nuclear-retained ncRNA known as mrhl is processed by Drosha to yield an 80-nt small RNA.
(C) Athough miRNAs and piwi-interfering RNAs (piRNAs) have received the most attention of late, that long RNA transcripts have important role in regulating the processing to small RNAs with likely different and unique functions.
(D) Long ncRNAs can be processed to yield small RNAs, but they can also affect how other transcripts are processed; for example, by modulating their ability to be cut into small RNAs or changing their pre-mRNA splicing patterns.
(E) ncRNA can inhibits the production of small RNAs from other transcripts.
(F) Non-Coding RNAs and Hormone regulation is an emerging field.